The present invention relates to the compound benziso-N(L-histidine methylester)-thiazolone of the formula 3 and a process for the preparation thereof. More particularly, the present invention also relates to the use of compound of formula 3 for RNA polymerase inhibition by zinc ejection from [CCXX] boxes (C=cysteine, X=cysteine or histidine). 
The development of molecular systems, which can inhibit enzymes upon binding or by the removal of metal ions from the active sites, or similar pathways, have been recognized as an important strategy towards better drug candidates. Zinc plays a very important role in many enzymes/proteins, either by imparting stability to the conformation required for the biological function or by taking part in the catalytic process. Reference may be made to Lynne Regan (Tibs, 1995, 281-285) and J. M. Berg et.al (J. Am. Chem. Soc., 1996, 118, 6514-6515), wherein the authors have found that the engineering of zinc finger domains in proteins could be used as a strategy for the modulation of its function and very recently, this approach has got enormous attention as a therapeutic tool for enzyme inhibition. Recent efforts in this domain have resulted in the identification of various reagents for zinc ejection from [CCXX] boxes, of which C-nitroso compounds (Rice, W. G.; Schaeffer, C. A.; Harten, B.; Villinger, F., South, T. L.; Summers, M. F.; Henderson, L. E.; Bess, J. W.; Arthur, L. O.; McDougall, J. S.; Orloff, S. L.; Mendeleyev, J.; Kun, E., Nature, 1993, 361, 473-475) and dithiobisbenzamides have got widespread recognition. The efficiency of dithiobisbenzamides in zinc ejection from nucleocapsid protein (NCp7) of HIV-1 has been studied in detail by Rice et. al. (Domagala, J. M.; Bader, J. P.; Gogliotti, R. D.; Sanchez, J. P. Stier, M. A.; Song, Y. Vara Prasad, J. V. N.; Tummino, P. J.; Scholten, J.; Harvey, P.; Holler, T., Gracheck, S.; Hupe, D.; Rice, W. G.; Schultz, R. Bioorganic and Medicinal Chemistry, 1997, 5, 3, 569-579).
The drawback in the above-referred work is the disproportionation of dithiobisbenzamides under the biological conditions, which may give problems in developing these as ideal inhibitors. These chelators generally lack target specificity, which limits their applicability towards a specific zinc finger domain.
The main objective of the present invention is to introduce Benziso-N(L-histidine methylester)-thiazolone of the formula (3) as a new candidate for RNA polymerase inhibition by zinc ejection from [CCXX] boxes, which is synthesized as per the scheme shown in FIG. 1. The carboxylic group present in 3 (as ester) could be used for anchoring various recognition elements for increasing the target specificity.
Another objective of the present invention is to highlight the importance of the exchange mechanism depicted in FIG. 2 during the chelation process, which is supported by the high activity of this compound compared to the traditional chelators like ethylenediamine tetraacetic acid or o-phenanthroline.
An examination of the zinc finger domains present in proteins show the involvement of cysteine and histidine residues in the coordination process. This suggested a biomimetic approach in reagent design by crafting a histidine composite system with reducible Sxe2x80x94N bond. We have successfully synthesized such a composite of formula (3) and demonstrated its efficiency in removing zinc by novel pathways envisaged in FIG. 2.
Recent advances in gene therapy necessitate the development of various agents that can interfere with information-function system at various levels. In this context, RNA polymerase, having subunit composition xcex12xcex2xcex2xe2x80x2"sgr" was selected for the exchange studies, because of its direct involvement in catalyzing transcription processxe2x80x94the first step in gene expression. Its xcex2xe2x80x2 subunit is known to posses two zinc finger domains. The N-terminal domain is somewhat buried and metal ejection is not effective under normal conditions. Previous studies have shown that zinc present at the C-terminal location can be removed or exchanged readily, leading to a modified enzyme, which showed much reduced activity (Markov, D.; Naryshkina, T., Mustaev, A. and Severinov, K. Genes and Development, 1999, 13, 2439-2448.)
Our studies have shown that dithiobisbenzamides derived from amino acids having a proton source in the side chain (Serine, Threonine, Tryptophan, Histidine etc.) have a high tendency to be transformed to benzisothiazolones, compared to those with neutral pendant groups (eg. Alanine, Valine, etc.). Thus, reaction of dithiodibenzoyl chloride with Histidine methylester gave benzisothiazolone 3 as the sole product (FIG. 1) in 56% yield. It is proposed that the thiophenol liberated is oxidatively recycled to the dithiobisbenzamide, which would account for the observed yield, which otherwise would have been xe2x89xa650%.
In a typical experiment, the course of transcription of calf thymus DNA by E. coli RNA polymerase was monitored in the presence and absence of the redox system 3. The enzyme aliquot after admixing with 3 was incubated for 15 minutes and then dialyzed overnight. A general transcription assay was then carried out in a medium, which contained 3H UTP in addition to other nucleotides. The amount of radioactive UTP (uridine triphaspate) incorporated in the transcribed RNA was monitored by scintillation counter and the count, when compared to that obtained on using native enzyme gave the percentage loss of activity.